Rheumatoid arthritis (RA) is an autoimmune disease in which T-cell activation plays a pivotal role in the induction of articular damage. CD4+/OX40+ T cells accumulate in the synovial fluid (SF) of RA patients, which suggests that they are involved in the pathogenesis of the disease. In this study, we assessed the intracellular cytokine production of peripheral blood and SF CD4+ and CD4+/OX40+ T cells from RA patients in order to evaluate their role in this disorder. Our results show that SF CD4+ cells are predominantly interferon gamma (IFN-gamma)-positive and express a Th1-like cytokine pattern. In SF, significantly more CD4+/OX40+ T cells expressed interleukin-4 (IL-4) and IL4/IFN-gamma than IFN-gamma alone. Our data demonstrate that SF CD4+/OX40+ T cells express a Th2/Th0 cytokine profile, which suggests that they are involved in inflammatory responses in RA joints.

We assessed the in vitro effects of interferon beta-1b (IFNbeta-1b), cyclophosphamide (CY), and azathioprine (AZA) alone and of the combination of IFNbeta-1b with CY or AZA on the production of Th1 and Th2 cytokines in 10 patients with multiple sclerosis. Cytokine levels were determined at baseline and after stimulation with IFNbeta-1b, CY, and AZA alone or with the combination of IFNbeta-1b with CY or AZA. The combination of IFNbeta-1b with CY resulted in a statistically significant decrease in the production of interleukin-2 (IL-2) (P=0.003) and tumor necrosis factor alpha (TNF-alpha) (P=0.03). An additive effect on the production of interferon gamma (IFN-gamma) (P=0.2) and interleukin-10 (IL-10) (P=0.6), and a positive interaction on the production of interleukin-4 (IL-4) (P=0.08) were observed although the findings were not statistically significant. The combination of IFNbeta-1b with AZA resulted in a significant negative effect on the production of IL-2 (P=0.006), whereas TNF-alpha (P=0.02), IFN-gamma (P=0.03), IL-4 (P=0.2), and IL-10 (P=0.3) were not statistically impacted. Our data show that CY was able to improve the effects of IFNbeta-1b on the ratio of Th1/Th2 cytokines.

Objective. To assess the percentage of T lymphocytes, bearing CD134. a member of the TNF receptor superfamily primarily found on autoreactive CD4+ T cells in the peripheral blood (PB) and synovial fluid (SF) of rheutmatoid arthritis (RA) patients.Methods. The surface expression of CD134 on SF and PB mononuclear cells was performed by flow cytometry in 25 RA patients and correlated to the disease activin:Results. CD134 expression on CD3+, CD4+, CD8+ and CD25+ cells was higher in SF than in PB of RA patients (P < 0.001). No differences were observed in the percentage of CD134+/CD4+ T lymphocytes in the PB of RA patients and controls. Patients with active RA had significantly higher percentage of CD3+/CD134+, CD4+/CD 134+, CD8+/CD134+ and CD25+/CD 134+ than those with inactive disease.Conclusion. These findings suggest that CD134+ T cells are involved in the immunopathological process of RA synovitis, maybe mirroring some other autoimmune disease in which autoreactive T cell infiltrating the target tissues largely coexpress CD134.

BACKGROUND AND AIM:The treatment of ulcerative colitis (UC) with 5-aminosalicylic acid (5-ASA) does not have the same therapeutic effect in all patients. We tested the hypothesis that the effectiveness of the drug is related to its mucosal concentration.PATIENTS:Twenty one UC patients receiving oral 5-ASA (2.4-3.2 g/day) were enrolled in the study. Four were also receiving topical treatment (2 g/day).METHODS:Six endoscopic biopsies were taken from the rectum for measurement of 5-ASA concentrations (ng/mg) by HPLC; soluble interleukin 2 receptor (sIL-2R) concentrations (U/ml) were measured by ELISA and histology. Endoscopic and histological appearance was graded on a four point scale (0-3). The Wilcoxon's rank test and Pearson's correlation coefficient were used for statistical analysis.RESULTS:Mucosal concentrations of 5-ASA were significantly higher (p=0.03) in patients with endoscopic scores of 0-1 compared with those with scores of 2-3 (16.1 (range 10.2-45) v 5. 5 (3.5-17.4), respectively) and in patients with lower histological inflammation compared with those with more severe scores (17.4 (10. 5-45) v 8.9 (3.5-17.2), respectively) (p<0.01). In contrast, mucosal sIL2-R concentrations were significantly lower in patients with slight endoscopic and histological lesions than in those with more severe disease. A significative inverse correlation (r=-0.85) was found between 5-ASA and sIL-2R mucosal concentrations (p=0.00008).CONCLUSIONS:In patients with UC, in the same area of the intestinal tract, we found that the higher the 5-ASA mucosal concentrations, the lower the IL-2R levels and endoscopic and histological scores. We hypothesise that maintenance of high mucosal 5-ASA concentrations in all colonic segments could contribute to improve clinical outcome in UC patients.

This study was performed in order to assess the cytotoxic activity, both natural (NK) and antibody-dependent (ADCC), of PBMC from 38 IBD patients and correlate it with their clinical features. Cytotoxicity assays were performed using sensitive target cells for NK and ADCC activities. In some experiments, highly purified NK cells, obtained both by Percoll density gradient and by co-culturing non-adherent PBMC with RPMI 8866 feeder cells, were used as effector cells. Furthermore, we evaluated NK cell parameters such as number, surface expression of adhesion molecules (CD11a/ CD18, CD49d and CD54) and response to different stimuli. We observed a decreased NK cytotoxicity of PBMC from IBD patients, both in ulcerative colitis (UC) and Crohn's disease (CD), independently of the clinical activity of disease. In contrast, the ADCC lytic activity was within normal range. The lower NK cytotoxic activity observed in our IBD patients cannot be related to a decreased number of NK cells, surface expression of adhesion molecules, defective response to IL-2 and maturative defect. Decreased NK activity was induced in PBMC of controls when serum of patients was added and this was unrelated to monocyte-derived modulating factor(s). Our data show a decreased natural killing by fresh PBMC from IBD patients. This lower activity seems to be unrelated to a primary NK cell defect, since purified NK cells exhibited normal levels of killing. It might be hypothesized that serum factors, possibly derived from lymphocytes, with inhibitory properties on NK activity, might be functionally active in the blood of IBD patients, thus modulating NK activity.

Imbalance in Th1 and Th2 subsets and their derived cytokines seems to be involved in the immune abnormalities underlying UC and CD. CD30 is a member of the tumour necrosis factor/nerve growth receptor superfamily expressed on T cells producing Th2 cytokines and released as a soluble form. In this study high levels of soluble CD30 were found in sera of UC patients independently of disease activity. Furthermore, increased titres of soluble CD30 molecule were shown, in the same patients, by mitogen-stimulated cultures of peripheral blood mononuclear cells. Our data seem to indicate that an activation of Th2 immune response is involved in the pathogenesis of UC, but not of CD. Furthermore, this finding indicates that serum soluble CD30 measurement may be helpful for differentiating these two forms of inflammatory bowel disease.

Activated Th2 lymphocytes express the surface molecule CD30 and release a soluble form of the same molecule which can be detected both in vivo and in vitro. In the present study, high levels of soluble CD30 were found in the peripheral blood of patients with SSc, and a significant correlation with skin score and erythrocyte sedimentation rate (ESR) was detected. Furthermore, we observed a higher spontaneous release of soluble CD30 in the supernatants of unstimulated cultures of peripheral blood mononuclear cells from our patients compared with healthy controls. Taken together, these data suggest a possible involvement of Th2 cells in the immunopathogenesis of SSc, and the dosage of CD30 soluble in the peripheral blood may be helpful in following the outcome of the disease.

In a previous study, we showed an imbalance in the cytotoxic phenotype of circulating PBMC of IBD patients, possibly related to an alteration in the cytotoxic activity, which might play a role in the immunopathogenesis of IBD. γ/δ T cells, which are increased in the PBMC of IBD patients, represent a minor population of peripheral lymphocytes displaying cytolytic potential and showing both MHC and non-MHC cytotoxicity. This study was performed in order to verify their role in the cytotoxic activity of PBMC from IBD patients and correlate this activity to UC and CD, respectively. We observed a decreased NK cytotoxicity of PBMC in both UC and CD patients and this findings was unrelated to γ/δ T lymphocytes. In fact, both total and γ/δ-depleted PBMC of IBD patients, showed comparable lytic activity. On the contrary, the ADCC lytic activity was within normal range in our patients, ad was not modified by removal of γ/δ cells. The increase of Vδ1+ T cells, previously observed in our patients, seems to be unrelated to this functional impairment, because of a low cytotoxic activity displayed by this subset. Alternatively, the expanded Vδ1+ T cells could be involved in the pathogenesis of the autoimmune phenomena observed in IBD, by a mechanism different from cytotoxicity, such as autoantibodies production and/or loss of tolerance.

OBJECTIVE To assess the ability of peripheral blood mononuclear cells (PBMC) of patients with systemic sclerosis (SSc) to produce interleukin 6 (IL-6), transforming growth factor beta 1 (TGF-beta 1), to identify the IL-6 producer cells in the in vitro model, and to correlate these data with the clinical evidence of our patients. METHODS We used a sandwich ELISA to quantitate IL-6 and TGF-beta 1 levels in sera, plasma, and supernatants, and an imunofluorescence technique to evaluate IL-6 producing cells in our patients. RESULTS IL-6 was detected in sera from 8 of 20 patients and no controls (p < 0.05). A significant increase of IL-6 production was observed in both spontaneous and phytohemagglutinin (PHA) induced cultures of PBMC from patients with SSc vs controls. No differences in TGF-beta 1 production were observed, either in sera or supernatants, between patients and controls. A significant increase of IL-6 synthesizing cells was observed after 3 h of PHA stimulation in patients vs controls (p < 0.05). CONCLUSION Spontaneous IL-6 production and the higher number of IL-6 producing cells in patients with SSc suggest that these cells have been already primed in vivo. The absence of PBMC primed for TGF-beta 1 production supports the hypothesis that cells other than lymphocytes produce and secrete this cytokine in the skin of patients. Higher serum levels of IL-6 observed in a subset of patients did not correlate with either severity or duration of disease.

In order to study the role of gamma/delta T cells in the pathogenesis of inflammatory bowel disease (IBD) in humans, we measured the percentage of these cells in the peripheral blood, assessed the ratio of the non-disulphide-linked (delta TCS1) type of T cell receptor (TCR) in the total gamma/delta T cells, studied the co-expression of gamma/delta TCR and accessory molecules CD8 and CD16, and compared these data with both the type and the activity of the disease. Percentage levels and absolute numbers of gamma/delta+ T cells were higher in active patients than in controls (P < 0.05), mainly as a result of an increase of V delta 1+ (delta TCS1) T cell subset (P < 0.05). This trend was strongly retained independently of disease activity and clinical picture. An increased percentage of TCR delta 1+/CD16+ cells was observed in our patients compared with controls (P < 0.05). In contrast, no difference was observed as far as the TCR delta 1+/CD8+ cells were concerned. These results suggest that IBD is associated with an expansion of gamma/delta T cells in peripheral blood, which may play a role in the pathogenesis of these disorders.